Skeletal myosin exists as unique isoforms in various muscles of the adult chicken and within the same skeletal muscle at different developmental ages. These myosin isozymes are characterized by their (1) unique mobilities during non-denaturing gel electrophoresis; (2) light chain composition. and (3) unique myosin heavy chain ad demonstated by peptide map analysis. In this proposal a combination of biochemical and immunological techniques will be employed to identify and characterize (1) myosin isoforms of fast and slow muscles of the developing white leghorn chicken and (2) myosin isoforms synthesized by myogenic cultures derived from fast and slow muscles of embryonic and adult chickens. Two separate approaches will be employed. Conventional antisera to 7 different myosin heavy chain isoforms have already been prepared. By employing absorption methods these sera will be made monospecific for the original antigen and using immunoflourescence techniques the myosin isoform compositions of individual muscle fibers of fast and slow muscles will be determined. A new technique is proposed combining peptide map analysis and immunoblotting using monospecific antibodies to produce "immunofingerprints" unique to each myosin heavy chain. The second approach will be to generate monoclonal antibodies to various myosin heavy chain isoforms using similar techniques that were successful in producing conventional rabbit antisera. The monoclonal antibodies demonstrating unique specificities will be used to (1) determine the isoform composition of individual muscle fibers in vivo and in vitro by immunoflourescence and (2) to map unique antigenic determinants on the native molecule and on the peptide map patterns of different myosin heavy chains. Those monoclona antibodies showing little or no specifity will be used to map common determinants in order to understand the structural relatedness of the various myosin isoforms. These studies will be important in understanding the regulation of muscle development and maturation.